Light emitting microorganisms and cells for diagnosis and therapy of diseases associated with wounded or inflamed tissue

ABSTRACT

Described is the use of a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, e.g., a luminescent or fluorescent protein for the preparation of a diagnostic composition for diagnosis and/or visualization of wounded or inflamed tissue or a disease associated therewith. Moreover, therapeutic uses are described, wherein said microorganism or cell additionally contain an expressible DNA sequence encoding a protein suitable for therapy, e.g. an enzyme causing cell death or digestion of debris.

The present invention relates to the use of a microorganism or cellcontaining a DNA sequence encoding a detectable protein or a proteincapable of inducing a detectable signal, e.g. a luminescent orfluorescent protein, for the preparation of a diagnostic composition fordiagnosis and/or visualization of wounded or inflamed tissue or adisease associated therewith. The present invention also relates totherapeutic uses wherein said microorganism or cell additionally containan expressible DNA sequence encoding a protein suitable for therapy,e.g. an enzyme causing cell death or digestion of debris.

Bacteremias may arise from traumatic injuries and surgical procedures aswell as from physiological functions, such as chewing or tooth brushing.Blood cultures taken before and after invasive procedures andphysiological functions from healthy human subjects show that while thepremanipulation blood samples are sterile, bacteria are present in theblood in varying frequencies depending on the procedures. A potentialconsequence of bacteremia is colonization of susceptible sites. However,despite the occurrence of transient bacteremias, only a certainpercentage of high-risk patients develop bacterial colonization ofpotentially susceptible sites. A number of investigators have suggestedthat bacteria from the blood circulation can colonize inflamed tissuesin animal models and on the surface of implanted materials. Theinconsistency in the pathological changes in humans following abacteremia may also be due to the resistance of host immune system, thevariability in the concentration of bacteria in the blood subsequent todifferent bacteremia events, and the virulence of any given bacterialstrain.

A number of investigators have focused on the nature of the implantedmaterials as the factor that influences the ability of bacteria toadhere. Materials such as sutures and surgical clips which are used forclosure of wounds, are potential sites of bacterial colonization.Infection of these materials may impede wound healing and/or placepatients at increased risk of secondary infections. A variety of woundclosure materials have been manufactured with varying affinities forbacteria. Certain wound closure materials, such as braided sutures, havebeen associated with a higher incidence of infection. The multifilamentnature of this type of suture material lends itself to increasedsusceptibility to bacterial colonization as well as causing a wickingeffect that allows penetration of bacteria across the tissues. Merepermanent implantable materials have demonstrated a similar affinity forbacteria. Prosthetic heart valves and joints may be at increased risk ofbacterial colonization. It is commonly believed that this highersusceptibility is caused by the inherent ability of bacteria to adheremore readily to the implant surfaces. An alternative explanation may bethat inflammation in the tissues surrounding the implants provides anenvironment that is more suitable for bacterial colonization. Inaddition to these given possibilities, another factor that may influencethe susceptibility of a site, with regards to colonization with bacteriacould be the degree of inflammatory status of the affected tissues.Implanted materials may create transient or chronic sites ofinflammation in the body.

Presence of implanted materials is not a requirement for bacterialcolonization. Alteration of natural anatomical structures that may arisefrom disease conditions may produce surfaces that are easier to colonizeby bacteria. It had been suggested that for the occurrence of infectiveendocarditis (IE), the valve surface must be altered in order to producea suitable site for bacterial attachment and colonization. Additionally,the microorganisms have to reach this site and adhere, since it is notpossible to produce IE in experimental animals with injections ofbacteria unless the valvular surface is damaged. Lesions with highturbulence create conditions that lead to bacterial colonization,whereas defects with a large surface area or low flow are seldomimplicated in IE.

However, so far, it could not be proven that transient bacteremiasactually cause colonization of inflamed or wounded tissue, since therewas no model available allowing the tracing of bacteria in a livingorganism, i.e. allowing to explain the temporal and spatial relationshipbetween bacterial infections and diseased tissue sites. Moreover,unfortunately, so far the early diagnosis and therapy of inflamed orwounded tissues or diseases associated therewith, e.g., anatherosclerotic disease, endocarditis, pericarditis etc., areunsatisfactory.

Therefore, it is the object of the present invention to provide a meansfor the efficient and reliable diagnosis as well as the therapy ofwounded or inflamed tissue or a disease associated therewith whichovercomes the disadvantages of the diagnostic and therapeutic approachespresently used.

According to the present invention this is achieved by the subjectmatters defined in the claims. In the experiments leading to the presentinvention it has been found that inflamed tissues, e.g. near implantedmaterial, permit bacterial colonization. Therefore, it is generallypossible to visualize inflamed tissues through use of the system of thepresent invention described below. It could be shown that expression ofgenes encoding light-emitting proteins in bacteria provides a genetictool that allows the tracing of the bacteria in a living host, i.e. theevaluation of the dynamics of an infection process in a living host. Theexternal detection of light-emitting bacteria allowed the inventors tonon-invasively study the spatial and temporal relationships betweeninfections and the manifested disease conditions. For generation of thelight-emitting bacteria, the bacterial luxab operon was used whichencodes the enzyme luciferase which catalyzes the oxidation of reducedflavin mononucleotide (FMNE2), in the presence of the substrate,decanal. This reaction then yields FMN, decanoic acid, water and aphoton of light. The light photons can then be captured by radiographs,luminometers, or by low light imagers. Recently, the entire bacterialluxcdabe operon, which encodes the substrate as well as the luciferaseenzyme, has been used for detection of bacteria in living animals. Theadvantage of this system is that it does not require exogenously addedsubstrate, which makes it ideal for in vivo studies.

In the studies leading to the present invention, the colonization ofwounded and inflamed tissue by bacteria initially present in thecirculating blood could be demonstrated and it could be shown thattissues that are irritated by implanted materials such as sutures, woundclosure clips and prosthetic devices are more susceptible to bacterialcolonization subsequent to bacteremias. The data obtained fromexperiments with the attenuated S. typhimurium shows that following anintravenous injection, bacteria disseminate throughout the body of thelive animals. Therefore, it is reasonable to suggest that the bacteriareach the wounded or inflamed sites via the circulation. These findingsdescribed in detail in the examples, below, open the way for (a)designing multifunctional viral vectors useful for the detection ofwounded or inflamed tissue based on signals like light emission orsignals that can be visualised by MRI and (b) the development ofbacterium- and mammalian cell-based wounded or inflamed tissue targetingsystems in combination with therapeutic gene constructs for thetreatment of diseases associated with wounded or inflamed tissue suchas, e.g., an atherosclerotic disease. These systems have the followingadvantages: (a) They target the wounded or inflamed tissue specificallywithout affecting normal tissue; (b) the expression and secretion of thetherapeutic gene constructs are, preferably, under the control of aninducible promoter, enabling secretion to be switched on or off; and (c)the location of the delivery system inside the tissue can be verified bydirect visualisation before activating gene expression and proteindelivery. Finally, there are a number of diagnostic methods that couldbe enhanced or advantageously replaced by the diagnostic approach of thepresent invention. For example, conventional angiography and MRAtechniques and MRA techniques both image blood flowing through the lumenof a vessel to visualize plaque, rather than imaging the plaquedirectly. MRA is particularly sensitive to turbulence caused by theplaque and, as a result, is often inaccurate. These shortcomings can beovercome by the diagnostic uses of the present invention.

Accordingly, the present invention relates to the use of a microorganismor cell containing a DNA sequence encoding a detectable protein or aprotein capable of inducing a detectable signal for the preparation of adiagnostic composition for diagnosis and/or visualization of wounded orinflamed tissue or a disease associated therewith. In addition, saidmicroorganism is also useful for therapy, since following visualizationof wounded or inflamed tissue compounds suitable for therapy can beapplied, e.g. by topical administration, such as, e.g., acylated iridoidglycosides from Scrophularia nodosa, cortisol, corticosteroid analogs,colchicine, methotrexate, non-steroidal anti-inflammatory drugs(NSAIDs), leflunomide, etanercept, minocycline, cyclosporine,thalidomide, a cytotoxic agent, 6-mercaptopurine, azathioprine,antibiotics or one or more of the proteins listed below.

The present invention also relates to the use of a microorganism or cellcontaining a DNA sequence encoding a detectable protein or a proteincapable of inducing a detectable signal for the preparation of apharmaceutical composition for the treatment of wounded or inflamedtissue or a disease associated therewith, wherein said micoroorganism orcell furthermore contains one or more expressible DNA sequences encoding(a) proteine(s) suitable for the therapy of wounded or inflamed tissueor diseases associated therewith.

Proteins suitable for the therapy of wounded or inflamed tissue ordiseases associated therewith include transforming growth factor(TGF-alpha), platelet-derived growth factor (PDG-F), keratinocyte growthfactor (KGF) and insulin-like growth factor-1 (IGF-1), insulin-likegrowth factor-binding proteins (IGFBPs), IL-4, IL-8, endothelin-1(ET-1), connective tissue growth factor (CTGF), TNF-alpha, vascularendothelial growth factor (VEGF), cyclooxygenase, cyclooxygenase-2inhibitor, infliximab (a chimeric anti-TNF-alpha monoclonal antibody),IL-10, lipase, protease, lysozyme, pro-apoptotic factor, peroxisomeproliferator-activated receptor (PPAR) agonist etc.

Any microorganism or cell is useful for the diagnostic and therapeuticuses of the present invention, provided that it replicates in theorganism, is not pathogenic for the organism e.g. attenuated and, isrecognized by the immune system of the organism, etc. The terms“microorganism” and “cell” as used herein refer to microorganisms andcells which are per se not targeted to wounded or inflamed tissues (i.e.they cannot differentiate between wounded or inflamed tissues and thenon-wounded or non-inflamed counterpart tissues) since the results ofthe experiments leading to the present invention show thatmicroorganisms and cells accumulate in wounded or inflamed tissues dueto the fact that in this environment they are not exposed to attack bythe immune system of the host. The microorganisms and cells accumulatefor a specific time, e.g. 3 to 5 days, as long as thevascularization/lymphatic system has not been restored.

In a preferred embodiment, the microorganism or cell contains a DNAsequence encoding a luminescent and/or fluorescent protein. As usedherein, the term “DNA sequence encoding a luminescent or fluorescentprotein” also comprises a DNA sequence encoding a luminescent andfluorescent protein as fusion protein.

In an alternative preferred embodiment of the use of the presentinvention, the microorganism or cell contains a DNA sequence encoding aprotein capable of inducing a signal detectable by magnetic resonanceimaging (MRI), e.g. a metal binding protein. Furthermore, the proteincan bind a contrasting agent, chromophore, or a compound required forvisualization of tissues.

Suitable devices for analysing the localization or distribution ofluminescent and/or fluorescent proteins in a tissue are well known tothe person skilled in the art and, furthermore described in theliterature cited above as well as the examples, below.

Preferably, for transfecting the cells the DNA sequences encoding adetectable protein or a protein capable of inducing a detectable signal,e.g., a luminescent or fluorescent protein, are present in a vector oran expression vector. A person skilled in the art is familiar withexamples thereof. The DNA sequences can also be contained in arecombinant virus containing appropriate expression cassettes. Suitableviruses that may be used include baculovirus, vaccinia, sindbis virus,Sendai virus, adenovirus, an AAV virus or a parvovirus, such as MVM orH-1. The vector may also be a retrovirus, such as MoMULV, MoMuLV,HaMuSV, MuMTV, RSV or GaLV. For expression in mammals, a suitablepromoter is e.g. human cytomegalovirus “immediate early promoter”(pCMV). Furthermore, tissue and/or organ specific promoters are useful.Preferably, the DNA sequences encoding a detectable protein or a proteincapable of inducing a detectable signal are operatively linked with apromoter allowing high expression. Such promoters, e.g. induciblepromoters are well-known to the person skilled in the art.

For generating the above described DNA sequences and for constructingexpression vectors or viruses which contain said DNA sequences, it ispossible to use general methods known in the art. These methods includee.g. in vitro recombination techniques, synthetic methods and in vivorecombination methods as described in Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., for example. Methods oftransfecting cells, of phenotypically selecting transfectants and ofexpressing the DNA sequences by using the above described vectors areknown in the art.

The person skilled in the art knows DNA sequences encoding luminescentor fluorescent proteins that can be used for carrying out the presentinvention. During the past decade, the identification and isolation ofstructural genes encoding light-emitting proteins from bacterialluciferase from Vibrio harveyi (Belas et al., Science 218 (1982),791-793) and from Vibrio fischerii (Foran and Brown, Nucleic acids Reds.16 (1988), 177), firefly luciferase (de Wet et al., Mol. Cell. Biol. 7(1987), 725-737), aequorin from Aeguorea Victoria (Prasher et al.,Biochem. 26 (1987), 1326-1332), Renilla luciferase from Renillareniformis (Lorenz et al., PNAS USA 88 (1991), 4438-4442) and greenfluorescent protein from Aequorea Victoria (Prasher et al., Gene 111(1987), 229-233) have been described that allow the tracing of bacteriaor viruses based on light emission. Transformation and expression ofthese genes in bacteria allows detection of bacterial colonies with theaid of the low light imaging camera or individual bacteria under thefluorescent microscope (Engebrecht et al., Science 227 (1985),1345-1347; Legocki et al., PNAS 83 (1986), 9080-9084; Chalfie et al.,Science 263 (1994), 802-805).

Luciferase genes have been expressed in a variety of organisms. Promoteractivation based on light emission, using luxAB fused to the nitrogenasepromoter, was demonstrated in Rhizobia residing within the cytoplasm ofcells of infected root nodules by low light imaging (Legocki et al.,PNAS 83 (1986), 9080-9084; O'Kane et al., J. Plant Mol. Biol. 10 (1988),387-399). Fusion of the lux A and lux B genes resulted in a fullyfunctional luciferase protein (Escher et al., PNAS 86 (1989),6528-6532). This fusion gene (Fab2) was introduced into Bacillussubtilis and Bacillus megatherium under the xylose promoter and then fedinto insect larvae and was injected into the hemolymph of worms. Imagingof light emission was conducted using a low light video camera. Themovement and localization of pathogenic bacteria in transgenicarabidopsis plants, which carry the pathogen-activated PALpromoter-bacterial luciferase fusion gene construct, was demonstrated bylocalizing Pseudomonas or Ervinia spp. infection under the low lightimager as well as in tomato plant and stacks of potatoes (Giacomin andSzalay, Plant Sci. 116 (1996), 59-72).

Thus, in a more preferred embodiment, the luminescent or fluorescentprotein present in the above described microorganism or cell isluciferase, RFP or GFP.

All of the luciferases expressed in bacteria require exogenously addedsubstrates such as decanal or coelenterazine for light emission. Incontrast, while visualization of GFP fluorescence does not require asubstrate, an excitation light source is needed. More recently, the genecluster encoding the bacterial luciferase and the proteins for providingdecanal within the cell, which includes luxCDABE was isolated fromXenorhabdus luminescens (Meighen and Szittner, J. Bacteriol. 174 (1992),5371-5381) and Photobacterium leiognathi (Lee et al., Eur. J. Biochem.201 (1991), 161-167) and transferred into bacteria resulting incontinuous light emission independent of exogenously added substrate(Fernandez-Pinas and Wolk, Gene 150 (1994), 169-174). Bacteriacontaining the complete lux operon sequence, when injectedintraperitoneally, intramuscularly, or intravenously, allowed thevisualization and localization of bacteria in live mice indicating thatthe luciferase light emission can penetrate the tissues and can bedetected externally (Contag et al., Mol. Microbiol. 18 (1995), 593-603).

Thus, in an even more preferred embodiment, the microorganism or cellcontaining a DNA sequence encoding a luciferase additionally contains agene encoding a substrate for a luciferase.

Preferably, the microorganism is a bacterium. Particularly preferred isattenuated Salmonella thyphimurium, attenuated Vibrio cholerae,attenuated Listeria monocytogenes or E.coli.

Alternatively, viruses such as Vaccinia virus, AAV, a retrovirus etc.are also useful for the diagnostic and therapeutic uses of the presentinvention. Preferably, the virus is Vaccinia virus.

Preferably, the cell for the uses of the present invention is amammalian cell such as a stem cell which can be autologous orheterologous concerning the organism.

In a further preferred embodiment, the microorganism or cell useful inthe present invention contains a ruc-gfp expression cassette whichcontains the Renilla luciferase (ruc) and Aequorea gfp cDNA sequencesunder the control of a strong synthetic early/late (PE/L) promoter ofVaccinia or the luxCDABE cassette.

In a preferred use of the microorganisms and cells described above theprotein suitable for the therapy of diseases associated with wounded orinflamed tissue like atherosclerotic diasease is an enzyme causing celldeath or an enzyme causing the digestion of debris, e.g. in the interiorof an atherosclerotic plaque causing the plaque to collapse under theforce of the intraluminal blood pressure. Suitable enzymes include alipase, protease, lysozyme, proapoptotic factor, PPAR-agonist etc. Ifthe inflammatory component of atherosclerosis should be treated suitablecompounds are cortisol, corticosteroid analogs, cyclooxygenase andcyclooxygenase-2 inhibitors, colchicine, methotrexate, NSAIDs,leflunomide, etanercept, minocycline, cyclosporine, thalidomide,infliximab, IL-10, 6-mercaptopurine, azathioprine or a cytotoxic agent.Some of these compounds might be in the form of pro-drugs.

Accordingly, the protein expressed by a microorganism of the inventioncan be an enzyme converting an inactive substance (pro-drug)administered to the organism into an active substance.

Preferably, the gene encoding an enzyme as discussed above is directedby an inducible promoter additionally ensuring that, e.g., theconversion of the pro-drug into the active substance only occurs in thetarget tissue, e.g., an IPTG-, antibiotic-, heat-, pH-, light-, metal-,aerobic-, host cell-, drug-, cell cycle- or tissue specific-induciblepromoter. Moreover, the delivery system of the present invention evenallows the application of compounds which could so far not be used fortherapy due to their high toxicity when systemically applied or due tothe fact that they cannot be administered, e.g., intravenously insufficiently high dosages to achieve levels inside, e.g., sinuses,abscesses or across the blood brain barrier. Such compounds includethalidomide, cytotoxic drugs, antibiotics etc.

Furthermore, the microorganism or cell of the present invention cancontain a BAC (Bacterial Artificial Chromosome)or MAC (MammalianArtificial Chromosome) encoding several or all proteins of a specificpathway, e.g. woundhealing-pathway, such as TNF-alpha, COX-2, CTGF etc.Additionally, the cell can be a cyber cell or cyber virus encoding theseproteins.

For administration, the microorganisms or cells described above arepreferably combined with suitable pharmaceutical carriers. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Administration of themicroorganisms or cells may be effected by different ways, e.g. byintravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration. The preferred route of administration isintravenous injection. The route of administration, of course, dependson the nature of the tissue and the kind of microorganisms or cellscontained in the pharmaceutical composition. The dosage regimen will bedetermined by the attending physician and other clinical factors. As iswell known in the medical arts, dosages for any one patient depends onmany factors, including the patient's size, body surface area, age, sex,the particular compound to be administered, time and route ofadministration, the kind and localisation of the tissue, general healthand other drugs being administered concurrently.

A preferred therapeutical use is the preparation of a pharamaceuticalcomposition for the treatment of endocarditis, pericarditis,imflammatory bowel disease (e.g. Crohn's disease or Ulcerative colitis),low back pain (herniated nucleus pulposis), temporal arteritis,polyarteritis nodosa or an arthritic disease.

In the past few years, there has been many reports showing evidence forChlamydia pneumoniae, Heliobacter pylori, CMV, HSV and other infectiousagents inside atherosclerotic plaques. The presence of these infectiousagents within atherosclerotic plaque suggests that the interior of theplaque is a protected environment that permits replication, otherwisethese infectious agents would be cleared by the immune system. Moreover,there is considerable evidence that an inflammatory process is presentwithin the interior of atherosclerotic plaque. Accordingly, it isreasonable to assume that this disease can be diagnosed and treated bythe microorganisms or cells of the present invention that—afterintravenous injection—will penetrate into the atherosclerotic plaquewhere they start to replicate. After a suitable period of time, theplaque can be imaged using, e.g., light sensitive cameras or suitableMRI equipment. Further, said microorganisms or cells can additionallyproduce an enzyme, e.g. an enzyme as described above, resulting in theelimination of plaques. Thus, a further preferred use is the diagnosisand treatment of an atherosclerotic disease.

A further preferred use is the diagnosis and treatment of coronaryartery disease, peripheral vascular disease or cerebral artery disease.Therapeutic treatments according to the present invention might replacetreatments like balloon angioplasty, stent placement, coronary arterybypass graft, carotid endarterectomy, aorto-femoral bypass graft andother invasive procedures. Moreover, plaque in inaccessible regions,such as the basilar and middle cerebral arteries can be treated usingthe therapeutic approach of the present invention.

For the therapy of wounds, fractures, surgical incisions and burns themicroorganisms of the present invention are preferably combined withproteins like transforming growth factor (TGF-alpha), platelet-derivedgrowth factor (PDG-F), keratinocyte growth factor (KGF) and insulin-likegrowth factor-1 (IGF-1), insulin-like growth factor-binding proteins(IGFBPs), IL-4, IL-8, endothelin-1 (ET-1), connective tissue growthfactor (CTGF), TNF-alpha, vascular endothelial growth factor (VEGF),cyclooxygenase, cyclooxygenase-2 inhibitor, infliximab (a chimericanti-TNF-alpha monoclonal antibody), IL-10, lipase, protease, lysozyme,pro-apoptotic factor, peroxisome proliferator-activated receptor (PPAR)agonist (or contain expressible DNA-sequences encoding said proteins).For the treatment of infectious diseases, the microorganisms of thepresent invention are preferably applied in combination withantibiotics. For the treatment of auto-immune and inflammatory diseases,including reumathoid arthritis, inflammatory bowel disease and multiplesclerosis, the microorganisms of the present invention are preferablyapplied in combination with cortisol, corticosteroid analogs,cyclooxygenase and cyclooxygenase-2 inhibitors, colchicine,methotrexate, NSAIDs, leflunomide, etanercept, minocycline,cyclosporine, thalidomide, infliximab, IL-10, 6-mercaptopurine,azathioprine or a cytotoxic agent. For the therapy of diseases likeatherosclerosis, the microorganisms of the present invention arepreferably applied in combination with lipases, lysozymes, pro-apoptopicfactors, PPAR-agonists (or the corresponding DNA-sequences) or an agentlisted above with respect to the treatment of inflammatory diseases. Forthe treatment of Alzheimer's disease, the microorganisms of the presentinvention are preferably applied in combination with one or more agentslisted above with respect to auto-immune- or inflammatory diseases.

Finally, the above described microorganisms and cells are useful for (a)monitoring the efficacy of an antibiotic regimen, preferably based onlight extinction or (b) comparing the resistance of various sutures andimplantable materials to bacterial colonization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Visualization of Bacteria Intravenously Injected into Nude Mice

Nude mice were injected with 1×10⁷ attenuated Salmonella typhimurium (A)or 1×10⁷ attenuated Vibrio cholera (B). Both strains were transformedwith pLITE201 carrying the lux operon. Photon collection was for oneminute 20 min after bacterial injections.

FIG. 2: Visualization of S. typhimurium in the Same Animal Over a 5-DayObservation Period

Nude mice were injected with 1×10⁷ attenuated S. typhimurium. On thefirst observation period, bacteria were disseminated throughout the bodyof the animal (A). Two days later, bacteria were cleared from the animalwith the exception of the incision wound and the ear tag region asindicated by the arrows (B). On day 5, the animal had been able to clearthe organism from the wounded regions (C).

FIG. 3: Visualization of V. cholera in the Same Animal Over an 8-DayObservation Period

Nude mice were injected with 1×10⁷ attenuated V. cholera. On the firstobservation period, bacteria were visualized in the liver region of theanimal (A). Five days later, bacteria were cleared from the entireanimal with the exception of the incision wound as indicated by thearrows (B). On day 8, the animal had been able to clear the organismfrom the wound (C).

FIG. 4: Visualization of V. cholera in an Immunocompetent C57 Mouse

1×10⁷ attenuated V. cholera were intravenously injected into the animal.Light-emitting bacteria colonized the ear tag on the forth day afterbacterial injection (indicated by the white arrow).

FIG. 5: Visualization of Light Emitting Bacteria in the Liver of Rats

Sprague Dawley rats were intravenously injected with 1×10⁸ attenuated E.coli transformed with the plasmid DNA pLITE201. carrying the luxcdabeoperon. Photons were collected immediately after infection for oneminute under the low light imager (Night Owl). Light emitting bacteriawere visualized in the liver of the whole live animal.

FIG. 6: Colonization of Rat Hearts with Light Emitting Bacteria

Intravenous injection of the rats with 1×10⁸ attenuated E. colitransformed with the plasmid pLITE201 carrying the luxedabe operon didnot lead to colonization of the hearts of control animals, which had notbeen catheterized (A). Similar induction of bacteremias in ratscatheterized through the right carotid artery lead to the colonizationof the heart with light emitting bacteria (B).

FIG. 7: Detection of Residual Bacteria in the Organs of Rats

Three days following intravenous injection of the rats with 1×10⁸attenuated E. coli, the hearts, livers, and spleens were excised andcultured overnight. Light emitting bacteria were visualized under thelow light imager (Hamamatsu) in all specimens from the catheterized rats(A-C), while in the control animals, bacteria were detected in the liver(A) and spleen (B) but not the heart (C).

The present invention is explained by the following examples.

EXAMPLE 1 Materials and Methods

(A)Bacterial Strains

The strains used were a non-pathogenic laboratory strain Escherichiacoli, strain DH5α, attenuated Salmonella typhimurium (SL7207 hisG46,DEL407 [aroA544::Tn101] and attenuated Vibrio cholerae (Bengal 2 Serotyp0139, M010 DattRSI).

(B) Plasmid Constructs

The plasmid DNA pLITE201 containing the luxcdabe gene cassette wasobtained from Dr. F. Marines (Voisey and Marines, Biotech. 24 (1998)56-58).

(C) Recipient Animals

Five- to six-week-old male BALB/c nu/nu mice (25-30 g body weight) andSprague Dawley rats (300-325 g body weight) were purchased from Harlan(Frederick, Md., USA). CS7BL/6J mice were obtained from JacksonLaboratories (Bar Harbor, Me., USA). All animal experiments were carriedout in accordance with protocols approved by the Lorna Linda Universityanimal research committee. The animals containing recombinant DNAmaterials and attenuated pathogens were kept in the Loma LindaUniversity animal care facility at biosafety level two.

(D) Detection of Luminescence

Immediately before imaging, the animals were anesthetized withintraperitoneal injections of sodium pentobarbital (Nembutal® Sodiumsolution, Abbot Laboratories, North Chicago, Ill.; 60 mg/kg bodyweight). The animals were placed inside the dark box for photon countingand recording superimposed images (ARGUS 100 Low Light Imaging System,Hamamatsu, Hamamatsu, Japan and Night Owl, Berthold Technologies, GmbHand Co. KG, Bad Wildbad, Germany). Photon collection was for one minutefrom ventral and dorsal views of the animals. A photographic image wasthen recorded and the low light image was superimposed over thephotographic image to demonstrate the location of luminescent activity.

EXAMPLE 2 Colonization of Cutaneous Wounds by Intravenously InjectedLight Emitting Bacteria in Live Animals

To determine the fate of intravenously injected luminescent bacteria inthe animals, 1×10⁷ bacteria carrying the pLITE201 plasmid DNA in 50 μlwere injected into the left femoral vein of nude mice under anesthesia.To expose the femoral vein, a 1-cm incision was made with a surgicalblade. Following closure of the incision with 6-0 sutures, the mice weremonitored under the low light imager and photon emissions were collectedfor one minute. Imaging of each animal was repeated at various timeintervals to study the dissemination of the light-emitting bacteriathroughout the body of the animals. It was found that the distributionpattern of light emission following an intravenous injection of bacteriainto the mice was bacterial-strain-dependent. Injection of attenuated S.typhimurium caused wide dissemination of the bacteria throughout thebody of the animals (FIG. 1A). This pattern of distribution was visiblewithin 5 minutes after bacterial injection and continued to be detectedat the one-hour observation period. Injection of attenuated V. cholerainto the bloodstream, however, resulted in light emission that waslocalized to the liver within 5 minutes after bacterial injection andremained visible in the liver at the one-hour observation period (FIG1B).

The difference in the bacterial distribution patterns suggests adifference in the interaction of these strains with the host once insidethe animal. Imaging the same animals 48 h after bacterial injectionshowed that all of the detectable light emission from the earlier timehad diminished and was eliminated completely from the injected animalwith the exception of the inflamed wounded tissues such as the incisionwound and the ear tag region. Inflammation in these tissues wasidentified by their red and edematous appearance. Light emission wasdetected in the incision wound and/or in the inflamed ear tag region upto 5 to 8 days postinjection, which was confirmed by longer photoncollection times, i.e. 10 minutes (FIG. 2A-C and FIG. 3A-C). The absenceof light emission was not due to the loss of the plasmid DNA or thesilencing of gene expression in the bacteria. In other experiments lightemission in animals could be consistently detected for up to 50 days.Similar data were obtained in immunocompetent C57BU6J mice (FIG. 4),showing that these observations are not limited to animals with alteredimmune systems. Careful examination of individually excised organs aswell as blood samples from infected animals confirmed the absence ofluminescence in these normal uninjured tissues. Furthermore, theexperimental data demonstrated that colonization of the injured tissuesis a common occurrence in mice. Twenty-four of 29 incision wounds(82.8%) and 12 of 29 ear tags (41.4%) in the mice were colonized byintravenously injected bacteria. Wound colonization by intravenouslyinjected bacteria occurred following injection of V. cholera inconcentrations as low as 1×10⁵ bacterial cells.

EXAMPLE 3 Colonization of Catheterized Rat Hearts Subsequent to FemoralVein Injection of Light-emitting Bacteria

Surgical heart defects were created according to the procedurespreviously described (Santoro and Levison, Infect. Immun. 19(3)(1978),915-918; Overholser et al., J. Infect. Dis. 155(1)(1987), 107-112).Briefly, animals were anesthetized with sodium pentobarbital (60 mg/kgi.p.). A midline neck incision was made to expose the tight carotidartery. A polypropylene catheter was introduced and advanced untilresistance was met indicating insertion to the level of the aorticvalve. The catheter was then secured using a 10-0 suture (AROSurgicalInstrument Corporation, Japan) and the incision was closed using 4-0silk sutures (American Cyanamide Company, Wayne, N.J.). Placement of thecatheter causes irritation and subsequent inflammation of the aorticvalve (Santoro and Levison, 1978). Control animals did not undergo thecatheterization procedure. Bacteremias were induced by injection of1×10⁸ light-emitting bacterial cells of E. coli via the femoral vein.When observed immediately after infection under the low light imager,bacterial colonization was visible in the liver region (FIG. 5). Threedays later, while catheterized animals consistently demonstratedcolonization of the heart with light emitting bacteria, control animalsshowed no sign of light emission from the heart (FIG. 6). To determineif low and undetectable levels of bacteria were present in the tissues,the heart, liver and spleen were excised from each animal and culturedovernight. The livers and spleens of the rats, which are organs that aredirectly involved in bacterial clearance, in both groups showed presenceof light emitting bacteria. Strong light emission was detected in thecatheterized heart in contrast to the control heart, which had completeabsence of emitted light (FIG. 7). No bacteria were detected on thecultured catheters.

These findings indicate that while light-emitting bacteria injected intothe bloodstream via the femoral vein were cleared from normal tissues,injured or inflamed tissues in immunocompromised and immunocompetentanimals provided sites that continued to retain bacteria for an extendedperiod of time.

1. A method, comprising: administering to a subject for whom thepresence or absence of a wound, wounded tissue, inflamed tissue or adisease associated therewith is to be detected, a microorganism or celldetectable in the subject; and monitoring the subject for detection ofthe microorganism or cell.
 2. A method, comprising: administering to asubject having a wound, wounded tissue, inflamed tissue or a diseaseassociated therewith a microorganism or cell detectable in a subject,wherein said microorganism or cell contains one or more nucleic acidsequences encoding a protein(s) suitable for the therapy of a wound,wounded tissue, inflamed tissue or a disease associated therewith. 3.The method of claim 1, wherein the microorganism or cell contains anucleic acid encoding a luminescent or fluorescent protein.
 4. Themethod of claim 3, wherein said luminescent or fluorescent protein isluciferase, RFP or GFP.
 5. The method of claim 4, wherein saidmicroorganism or cell additionally contains a nucleic acid encoding asubstrate for a luciferase.
 6. The method of claim 1, wherein themicroorganism or cell contains a nucleic acid encoding a protein capableof inducing a signal detectable by magnetic resonance imaging (MRI) orcapable of binding a contrasting agent, chromophore or a ligand.
 7. Themethod of claim 1 wherein said microorganism is a bacterium or a virus.8. The method of claim 7, wherein said virus is Vaccinia virus.
 9. Themethod of claim 7, wherein said bacterium is selected from the groupconsisting of an attenuated Salmonella thyphimurium, an attenuatedVibrio cholerae, an attenuated Listeria monocytogenes and E. coli. 10.The method of claim 1, wherein the cell is a mammalian cell.
 11. Themethod of claim 10, wherein the mammalian cell is an autologous orheterologous stem cell.
 12. The method of claim 2, wherein said proteinsuitable for the therapy of a wound, wounded tissue, inflamed tissue ora disease associated therewith is an enzyme causing cell death or anenzyme causing the digestion of debris.
 13. The method of claim 2,wherein said disease is selected from the group consisting ofendocarditis, pericarditis, inflammatory bowel disease, low back pain(herniated nucleus pulposis), temporal arteritis, polyarteritis nodosaand an arthritic disease.
 14. The method of claim 2, wherein saiddisease is an atherosclerotic disease.
 15. The method of claim 2,wherein said disease is selected from the group consisting of coronaryartery disease, peripheral vascular disease and cerebral artery disease.16. The method of claim 1, wherein said monitoring is carried out byMRI.
 17. The method of claim 2, wherein said nucleic acid sequences areon a BAC, MAC, cyber cell or cyber virus.
 18. The method of claim 2,wherein said nucleic acid sequence is under the control of an induciblepromoter.
 19. A method comprising: administering to a subject who isbeing treated with an antibiotic, has been treated with sutures and/orhas been treated with an implantable material, a microorganism or celldetectable in the subject; and detecting the microorganism or cell to:(a) monitor the efficacy of an antibiotic regimen; (b) evaluate theresistance of a suture to bacterial colonization; and/or (c) evaluatethe resistance of an implantable material to bacterial colonization. 20.The method of claim 2, wherein the microorganism or cell replicates inthe subject, is not pathogenic to the subject and is recognized by theimmune system of the subject.